The Depression of the Synthesis of Pea Diamine Oxidase Due to Light and the Verification of Its Participation in Growth Processes Using Competitive Inhibitors

BIOLOGIA PLA_NTARD2r (PRAHA) 16 (2) : 86--93, 1974

The Depression of the Synthesis of Pea Diamine Oxidase due to Light and the Verification of its Participation in Growth Processes using Competitive inhibitors

L. MACHOLi~ and J. Mr~s

Departments of Biochemistry and Plant Biology, J. E. Purkynii University, Brno, Czechoslovakia*

Received January 2, 1973

Abstraet. The time courses of the synthesis of diamine oxidase in pea plants grown for 14 days either in the light or in the dark are similar with the highest increase in activity occurring in the cotyledons and in the shoots during the first 6 to 8 days. Plants grown in the dark showed a 2- to 3-fold higher enzyme activity than plants grown in the light. Pea diamine oxidase could be in rive efficiently inhibited by substrate analogues 1,4-diamino-2-butanono and 1,5-diamino-3-pentanone. The first compound inhibited proportionally to its concentration the growth of etiolated pea plants, but its instability makes an unoqulvocal interpretation of the results difficult. On the other hand, 1,5-diamino-3-pontanono a stable and more efficient diamino oxldaso inhibitor depressed the growth of pea seedlings only at concentrations as high as 5 m)5 and 10 raM, at which the growth of cress seedlings not containing diamine oxidaso was also strongly depressed. The results obtained indicate that tryptamine oxidation catalyzed by diamine oxidase is not involved in the main metabolic pathway leading from tryptophan to indoleacetate in pea plants.

Young pea plants contain high diamine oxidase activity [diamine: oxygen oxidoreduetase (deaminating), EC 1.4.3.6.) which is sometimes simply referred to as amine oxidase. The synthesis of the enzyme starts in the first hours of germination and in normal green plants reaches the highest activity after the first few days (K~TE~ and MA~ 1952, W~.~L~ et al. 1959, 1961). Because amine oxidases are able to oxidize tryptamine, some authors supposed that it could be involved in the formation of indoleacetate (KENTE~ and MA~ 1952, CL)_~KE and MA~ 1957, LAI~TICA~ and MUIR 1967). Ex- periments carried out so far mostly with the inhibitors of hydrazine type lead, however, to contradictory conclusions. REED et al. (1965) and recently KI~URA and TA~AK)~ (1971) found a relation between depressed growth of pea plants and the inhibition of tryptamine oxidation caused by the herbicide B-995 or by monoamine oxidase inhibitors safrazine and nialamide. On the

Address: Kotls 2, 611 37 Brno, Czechoslovakia.

86 SYNTHESIS OF PEA D~NE OXIDASE 87

contrary, R. Moss from the Gordon's laboratory (see LARSE~ 1967, page 433) found that indoleacotate production was not changed in the pea stem tissues in which tryptamine oxidase activity was nearly completely inhibited. Aminoguanidine, isonicotinio acid hydrazide, and isopropylhydrazino in- activating diamine oxidase activity in vivo inhibited the germination not only in pea but even in the plant species not containing this enzyme (WEI~LE et al. 1959). In this paper we therefore try to verify if the selective inhibition of diamine oxidase can influence the germination and growth of pea seedlings. Nearly untoxical substrate analogues 1,4-diamino-2-butanone and 1,5-diamino-3-pontanone were used as efficient competitive inhibitors of diamine oxidase; they do not affect mitochondrial monoamine oxidase (MAc~oLA~ et al. 1967, MACHOLA~r 1974). Another aim of this paper was to follow the dynamics of the formation of diamine oxidase in pea plants grown either in the light or in the dark. Material and Methods 1,4-diamino-2-butanone dihydrochlerido was prepared after MICltALSK~ etal. (1953) and 1,5-diamino-3-pentanone was synthesized according to a method devised in our laboratory (MAc~OL%_lq 1974).

Cultivation of the Plants The experiments were performed with pea seedlings (Pisum sativum L.) cv. Liliput, and, cross plants (Lepidium sativum L.) were used for bioassays. Selected seeds were surface sterilized for 5 rain. with 96% ethanol, rinsed with water, and then soaked for 24 h in distilled water or in inhibiter solution of pH approx. 5.0. The seeds were then transferred onto a layer of granulated polyethylene underlayed with diluted (1 : 4) Richtor's nutrient solution supplemented with microolements in Hoaglan4 A--Z solution. The germination and further cultivation occurred at 23 ~ either in the dark or in continuous light (white and rosy fluorescent tubes 1 : 1, 7 000 lx at the level of the plants) in a chamber with controlled conditions. The volume of the nutrient solution was kept constant by replenishing with distilled water. In the experiments with the inhibitors, groups of 250 seedlings, 3 to 4 days old, were transferred into 1 000 ml flat plastic cultivation vessels containing 500 ml of diluted (1 : 1) nutrient solution either with the inhibitor or without it.

Enzyme Extraction and Assays 10 to 22 pea seedlings were carefully washed, and then cotyledons, roots, and shoots wore sepa- rated and weighted. Tostao wore removed from the cotyledons and each plant part was separately homogenized in a mortar with chilled 0.1 M phosphate buffer pH 7.0 and sdica sand (0.2 g of sand per g of plant material). The homogonato was sqeeze4 through a nylon cloth and the remaining pellet was repeatedly extracted with the buffer solution. The filtrate was made up to 50 or 100 ml with the buffer solution and immediately assayed. Protein was determined in the supornatant after centrifuging the homogenate at 10 000 g for 15 rain u~ing a modifigd biurot method with the subtraction of the turbidity after the addition of KCN (SzARI~OWSKX and KLI~'GENBERG 1963). Bovine sorumalbumin was used as standard protein. Diamino oxidaue activity was determined spectrophotomotrically ([-IoI, MSTEDT etal. 1961, M~-C~OLs 1966) at 25 ~ The assay mixture (final volume 5 ml) consisted of 80 mM phosphate buffer pl-I 7.0, 2.5 mM c-aminobenzaldehyde, 20 ~g catalase, 10 mM 1,4-diaminobutano, and 0.5 ml plant extract. One unit (lJ) represents the amount of enzyme catalyzing the formation of one fxmole Al-pyrrolino per rain. Specific activity is expressed in units per mg protein.

Results

Diamine Oxhlase Level in Pea Plants Grown Either in the Light or in the Dark Diamine oxidase activity was followed in extracts from different parts of plants grown in continuous light or in the dark during the first 14 days. The 88 h. ~[ACHOL~i.N, J. MINAI~

COTYLEDONS SHOOT

DAB

o4d eed O~ o9e

D o

o I ~ I I /

DAB

oee oed D O11d

DAP

o Yd 10d o13d

01 , , ~ ,-- n 2 4 6 8 10 12 2 4 6 8 10 0 2 4 6 8 10 TIME [d] INHIBITOR CONCENTRATION [10 .3 M]

Fig. 1. Diamine oxidase activity in pea plants grown in the light and in the dark. Enzyme activity in the cotyledons (~), in the shoots @, and in the roots[~, as expressed per g fresh weight -- A, per plant -- B, and per mg soluble protein -- C. Full symbols: plants grown in the dark, open symbols: plants grown in the light. <1, Diamine oxidase activity in above-ground parts of etiolated plants which were transferred at the time denoted by an arrow into the continuous light. Fig. 2. The inhibition of diamine oxidase in the cotyledons and in the above-ground parts of pea plants grown in the dark by 1,4-diamino-2-butanone (DAB) and 1,5-diamino-3-pentanone (DAP). The inhibitor was applied in the concentrations given in the abscissa either only when soaking the seeds (A), or both to seeds and roots in the diluted (1 : 1) l~ichter's nutrient solution (B, C)o found values were expressed per g fresh weight (Fig. 1A), per plant (Fig. IB), or per mg total soluble protein (Fig. 1C). The synthesis of the enzyme starts soon after the swelling of the seeds and its activity rises sharply in the follow- ing 6 to 8 days, mainly in the cotyledons. This increase in activity is most conspicuous when the activity is expressed per plant. We can see that after an initial sharp increase, the activity changes only slowly in both normal and etiolated plants. When the activity was expressed per fresh weight unit a decrease in diamine oxidase activity was established in the above-ground SYNTHESIS OF PEA DIAMINE OXIDASE 89 parts and in the roots which was obviously connected with the growth of these organs. In the cotyledons which gradually loose their weight, the level of diamine oxidase continuously slowly rises. Specific activity of diamine oxidase progressively rises in the cotyledons but decreases after an initial increase in the above-ground parts during the development of the seedlings. The plants grown in the dark always showed a higher diamine oxidase activity then those grown in the light. At the end of the 14th day of cultivation approx, a twofold activity was found in the cotyledons and approx. a threefold activity was found in the above-ground parts when expressed per plant (Fig. 1B). When 10 days old etiolated plants were transferred into the light diamine oxidase activity remained at the same level as in control plants kept continuously in the dark (Fig. 1A). This indicates that only the initial phase of the synthesis of diamine oxidase is controlled by the light.

The Influence of the Inhibitors The competitive inhibitors of diamine oxidase, 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone, were tested on pea plants grown in water cultures in the dark. 1,4-diamino-2-butanone was applied at the beginning of the experiments to the soaking seeds in 0.625 to 10mM water solutions. Diamine oxidase activity was considerably decreased in comparison to control plants in the cotyledons and in the above-ground parts whereas it was totMy inhibited in the roots. However, as the growth progressed, diamine oxidaso activity increased in the above-ground parts and in the cotyledons (Fig. 2A). For this reason both inhibitors were added in further experiments to the nutrient medium as well and as a result a steady and more pronounced inhibition of diamine oxidase was achieved (Fig. 2B, 2C). In agreement with the established values of inhibition constants (MAc~OL~N 1974) 1,5-diamino- -3-pentanone proved to be a stronger in vivo inhibitor (Ki ~--- 1.5 • 10 -s M) than 1,4-diamino-2-butanone (Ki = 2.1• 10 .7 M). The 5 mM and 10 mM concentrations of 1,5-diamino-3-pentanone totaly inhibited diamine oxidase activity in all investigated plant organs (Fig. 2C). Neither of both inhibitors influenced the germination of pea seeds, but the growth of the seedlings was delayed mainly in the solutions with the highest inhibitor concentrations. 1,4-diamino-2-butanone. surprisingly inhibited the growth of the seedlings grown in the dark more than its higher homologue (Fig. 3A- 3C). We suppose that this was caused by its instability in neutral media (MAcHOLi~ et al. 1967). The nutrient solution with its addition, and further the roots, the cotyledons and the basal parts of the epicotyls of the plants grown in this nutrient solution turned dark owing to the spontaneous formation of decomposition products in the course of the cultivation. With the stable and more efficient inhibitor 1,5-diamino-3-pentanone, the growth of pea seedlings was inhibited only slightly even at the highest concentrations (Fig. 3C). Moreover, the inhibition of the growth is not specific and obviously is not in any connection with the inhibition of diamine oxidase, as the inhibitor explicitely inhibited in higher concentrations also the growth of the hypocotyls and roots of cress plants in which diamine oxidase was not found (Table 1). It is of interest that apical dominance was impaired by the inhibitors in some pea plants which resulted in the formation of lateral shoots 90 L. MACHOL~_N, J. MIN~

TABLE 1 THE EFFECT OF THE INHIBITORS OF DIA~IINE OXIDASE ON THE GROWTH OF CRESS PLANTS A~EI~ TWO DAYS OF CULTIVATION

Concentration Length [mm] Inhibitor [mM] Hypocotyl Root

h 23.50 -4- 0.76 32.03 4- 1.58 1,4-diamino- 0.625 23.73 ~: 0.83 30.35 • 1.32 2-butanone 1.25 20.23 -V 1.14" 28.13 -t- 1.33 2.50 18.24 4- 0.90** 20.70 4- 1.37"* 5.00 11.35 4- 0.71"* 8.28 4- 1.22"* 10.00 3.40 :L 0.34** 2.12 4- 0.06** 1,5-diamino- 0.625 24.28 4- 0.90 36.23 4- 1.52 3 -pentanone 1.25 20.18 4- 0.72** 28.85 + 1.15 2.50 12.05 ~: 0.62** 22.65 4- 1.82"* 5.00 5.15 ~ 0.49** 4.85 ~ 0.68** 10.00 2.35 4- 0.12"* 2.08 ~: 0.04**

The difference from the control (no inhibitor) is st;at~istically significant * ~P < 0.05; ** =P < 0.01; n= 40

from cotyledon axillary buds. With 10 mM 1,4-diamino-2-butanone 80% of treated plants formed lateral shoots in 20 days. To find whether applied inhibitors were transported up to the shoot apex, the above-ground parts of the pea plants grown in the dark were cut into four segments of equal length and then separately analyzed. In the control plants grown without the inhibitors, the highest diamine oxidase activity was found in the apical and especially in the basal segment which is in agreement with the findings of WERLE et al. (1959). In plants grown from the beginning with 5 mM inhibitor, diamine oxidase was inhibited in all segments (Table 2). The same occurred in the plants transferred to the solution with the inhibitor after seven days of normal cultivation. Thus, the inhibitors are transported from the roots to the whole plant including the cotyledons where diamine oxidase activity was completely depressed.

3C 14d ed 7d Fig. 3. The influence of competitive A ~6d B {~ 8d C [] 10d [~Sd [] 11d [] 13d inhibitors of diamino oxidase on the growth of above-ground parts of etiolated pea plants. A -- the grow E O F th of the seedlings from the seeds 2C soaked in the water solution of 2: I. I ~ ?1 1,4-diamino-2-butanone; B, C -- i i _L i the growth of the seedlings which .J were given 1,4-diamino-2.butanone ' rlI'l or 1,5-diamino-3-pentanono re- r~7 I rZ~ z spectively to the nutrient solution E (D in addition to the treatment of the seeds. Inhibitor concentration: 1 - 6.25 • 10 -4 M; 2 - 1.25 • 10 -3 M; 3-2.5• -3M~ 4--5.0• -3M; 5 -- 1 • 10 .3 M; 0 = control without i ~ili l i the inhibitor. Diamine oxidase in- I 2 3 4 5 012345 012 3 4 5 hibitiou is shown in the Fig. 2. INHIBITOR CONCENTRATION SYNTHESIS OF PEA DIAMINE OXIDASE 91

TAnLE 2

THE INHIBITIOI~ OF DIAMINE OXIDASE IN DIFFERENT SHOOT SEGI~ENTS OF ETIOLATED PEA PLANTS

Average Diamino oxidaso activity* length [mU per plant] The way of inhibitor application Inhibitor of the and of the cultivation (SmM) Segment ~o. (from the top) shoot [om] 1 2 3 4 total

The seeds wore soaked in inhibi- IIO 8.88 230 204 373 552 1359 tor solution and 4 clays old seed- DAP 5.38 19 18 14 16 67 lings were cultivar for 3 days DAB 6.30 36 25 15 13 89 in the nutrient solution with the inhibitor

The seeds wore soaked in distilled no 16.94 ~43 197 326 789 1555 water and 4 days old seedlings DAP 14.27 66 29 7 0 102 were cultivated for 3 clays in the -IDAB I 14.61 66 19 0 8 93 nutrient solution and for further 3 days in the nutrient solution with the inhibitor

* l~Iean value of 15 plants; DAP ~ 1,5-diamino-3-pontanone; DAB = 1,4-diamino-2-butanone

By means of dialysis it was proved that both inhibitors inactivate diamine oxidase but do not inhibit its synthesis. The experiments were carried out with the extracts from the cotyledons of the plants grown continuously in 5 to 10 mM diamino-ketone solutions in which no diamine oxidase activity could be found before the dialysis. 52 to 59% of activity (related to the control without the inhibitor) could be restored after 30 h of dialysis at 5 ~ against 50 mM phosphate buffer pH 7 in the case of 1,5-diamino-3-pentanone treatment, and approx. 40 ~ of activity in the case of 1,4-diamino-2-butanone treatment. The activity did not further increase with prolonged of dialysis, probably as a result of partial irreversible inhibition which takes place when diamine oxidase is exposed for a long time to an excess of a diaminoketone, especially in the case of 1,4-diamino-2-butanone (MAc~OLX~ 1974).

Discussion Purified pea diamine oxidase oxidizes in vitro tryptamine to indoleacet- aldehyde, which can be further oxidized by other enzyme systems occurring in crude pea extracts (CLARKE and MA~ 1957). The experiments described in this paper were to verify the significance of diamine oxidase for the growth of pea seedlings. The used competitive inhibitors inactivated the enzyme in vivo as was shown by the determination of enzyme activity in diluted homo- genates. The growth of the above-ground parts and roots was not depressed significantly even by relatively high concentrations of the stable 1,5-diamino- -3-pentanone even though diamine oxidase activity fell down in the whole plant to the threshold of sensitivity of the method used in the experiments described in this paper with 1,4-diaminobutane as the substrate. Tryptamine, the supposed auxin precursor, would be oxidized still 15 times slower under 92 L. I~[ACHOL~, J. 3~IN/klr% the conditions used for the assay (HILL and MANly 1964). Therefore, it can be deduced from the results that the inhibition of diamine oxidase activity does not lead to a pronounced reduction in auxin level, and that this enzyme is not involved in the main pathway leading to its biogenesis. Some recent in- vestigations have shown that pea plants do not contain tryptamine and are not able to synthetize it from tryptophan under normal conditions (LIBBERT 1967, KVTs and KEFELI 1970). Indoleacetate is also formed in crude enzyme preparations much slower from exogenous tryptamine than from tryptophan or indolepyruvate (LIBBERT and BRuN~ 1961). As the biosyn- thesis of indolepyruvate from tryptophan was also confirmed in the experiments with stem segments of aseptically grown pea plants (LIBBE~W 1967), it seems that the aminotransferase enzyme system is predominant in the synthesis of auxin in pea plants (cf. LARSE:h" 1967). The biological function of pea diamine oxidase should probably be looked for in the processes connected with the initial nutrition of the seedlings. At this stage diamine oxidase is formed in high concentration mainly in the cotyledons, and the stem stimulates significantly its formation (SuzuKI and YAMASAKI 1971). Plants grown in the dark limited in their nutrition to the cotyledon reserves have a clearly higher diamine oxidase activity than the plants grown in the light. Diamine oxidase is obviously metabolically considerably resistant owing to the fact that the decrease in the reserve content in the cotyledons its specific activity enormously increases. This "nature! form of enzyme purification" can be useful when isolating the enzyme.

Acknowledgement The skilled technical assistance of Miss d. HaubrovA and Mrs. M. KolAi'ovs is gratefully ac- knowledged. References CLARKE, A. J., MA~N, P. J. G.: The oxidation of tryptamine to 3-indolylaeetaldehyde by plant amino oxidase. -- Biochcm. J. 65 : 763--774, 1957. HILL, J. M., MXN~, P. J. G.: Further properties of the diamine oxidaso of pea seedlings. -- Biochem. J. 91 : 171--182, 1964. HOLMSTEDT, B., LARSSON, L., TttAM, R.: Further studies of a spectrophotometric method for the determination of diamine oxidase activity. -- Biochim. biophys. Acts 48 : 182--186, 1961. KENTEN, R. H., MANN, P. J. G. : The oxidation of amines by extracts of pea seedlings. -- Biochem. J. 50 : 360--369, 1952. KI~UI~A, 1VI., TXNAXA, Cm : Inhibitory effects of monoamine oxldase inhibitors on the growth of pea and rice seedlings. -- Plant Cell Physiol. 12 : 551--558, 1971. KUTX(~EK, M., I~EFELI, V.: Biogenesis of indole compounds from D- and L-tryptophan in segments of etiolated seedlings of cabbage, maize and pea. -- Biol. Plant. 12 : 145--158, 1970. LANTICAN, B. P., MUIR, 1=~. M.: Isolation and properties of the enzyme system forming indole- acetic acid. -- Plant Physiol. 42 : 1158--1160, 1967. LA~SEN, P. : The biogenesis ofindolo auxins. -- Wiss. Z. Univ. :Restock, Math.-Nat. Reihe 16 : 431 to 438, 1967. LmBERT, E.: Die Bedeutung epiphytischor Bakterien fiir den Auxinstoffwechsel hSherer Pflan- zon. -- Wiss. Z. Univ, Restock, Math.-Nat. Roihe 16 : 457--462, 1967. L1:BB]~RT, E., BRVN~, K. : Nachweis yon Indol-3-bronztrauhens~ure und Indol-3-/ithanol (Trypto- phol) bei der enzymatischen Auxinbildung aus Tryptophan in vitro. -i~aturwiss. 4B : 741, 1961. MAC~OL~N, L.: Boitrag zur spoktrophotomotrischon Bestimmung dor enzymatischen 0xydation ~,on aliphatischon Diaminen. -- Coll. czochoslov, chem. Commun. 31 : 2167--2174, 1966 SYNTHESIS OF PEA DIA1VIINE OXIDASE 93

M~0NOI~$,~, L.: Selective and reversible inhibition of diamine oxidase by 1,5-diamino-3-pentanone. Coll. czechoslov, chem. Commun. 39 : 653--661, 1974. M.Acztol,J,l~, L., ROZPI~IMOV~, L., SEDI,X~XOV.i, E.: 1,4-Diamino-2-butanon (2-ke~oputroseino) as strong and short acting competitive inLibitor of dmmine oxidase. -- Biochim. biophys. Acta 132 : 505--507, 1967. MICH&LSK'/:, J., BORKOVEC, J., I-LtD/~EK, J.: P~iprava 1,4-diamino-butanonu-(2). [Preparation of 1,4-diamino-2-butanone.] -- Chem. Listy (Praha) 47 : 1239--1241, 1953. REED, D. J., MOORE, T. C., ANDE~SOSr, J. D.: Plant growth retardant B-995: A possible mode of action. -- Science 148 : 1469--1471, 1965. SuzuKI, Y., YA-'CIASAKI,K.: Effects of some inhibitors of nucleic acid and protein synthesis on the development of amine ox~daso in germinating pea cotyledons. -- Physiol. Plant. 24 : 441 to 445, 1971. SZARKOWSKA, L., KLI~E~CB~I~a, M.: On the role of ubiquinono in mitochondria. Spectre- photometric and chemical measurements of its rodox reactions.- Biochom. Z. 338 : 674--697, 1963. WERLE, E., BEAUCA~P, G., SC~III~REN, V.: l~bor die Aminoxydaso yon Erbsonkeimlingon mid iht'e Bedoutung fiir die Koimung. -- Planta 53 : 125--133, 1959. WERLE, E., TR~VWSC~OI,D, I., AUI~ES, D. : Roinigung un4 Charakterisierung dot Diamin-Oxydase aus Erbson. -- Z. physiol. Chem. 326 : 200--211, 1961.

L. I~[ACHOL~Iq, ,1. ~][IN'~, Katodra biochemie a katedra biologle rosthn, pHrodov~deckg~ fa- kulta, Universita J. E. Purkyni~, Brno: Deprese tvorby hraehov6 dlaminoxid~izu vUvem svlitla a p~ezkoum~ini |eji role v rdstovSeh proeeseehpomoei kompetltivnieh inhibitorfi. -- Biol. Plant. 16 : 86--93, 1974. Tvorba 4iaminoxidhzy (EC 1.4.3.6) v hraehu, kultivovan6m pc dobu 14 dnfi na svittlo nebo ve trait, mt~ podobn~" prtkb~h s nojv~tw pHrfistky aktivity v d~loh~ch a nadzomnlch 6~stech asi do 6 a~ 8 dnfi rfistu. V etiolovan:~ch rostlingmh byla nam~ena v jednotliv~ch 6g~stech 2 • a~ 3 • vy~w enzymov~, a]